The detection of circulating fetal cells in maternal blood samples represents an important area of laboratory support to the obstetrical management of women. Although the concentration of fetal erythrocytes found in the maternal blood circulation during pregnancy is mostly very small and without a clear clinical significance in many cases, substantial hemorrhage may result from a number of causes including fetal or maternal trauma and placental defects (1). The quantification of fetal red blood cells (RBCs) is most commonly used to estimate the degree of feto-maternal hemorrhage (FMH), either in cases of trauma with suspected placental injury or in the situation of RhD incompatibility between the fetus and the mother for prevention of hemolytic disease of the newborn (HDN) during pregnancy (2, 3). The obstetrical management of women includes the prevention of mother immunization against a foreign fetal cell antigen and the monitoring of maternal antibody concentration. To prevent an immune response, an immunoprophylaxis based on anti-RhD polyclonal antibodies is given to the mother at a dose proportional to the estimated count of fetal RBCs present in the maternal blood circulation (4, 5). It is, therefore, important to be able to at least semi-quantify the relative amount of the cells.
Most clinical laboratories perform FMH estimates on the basis of variations of the slide-based microscopic counting method of acid elution originally described as the Kleihauer-Betke test (6). Although this assay has proven to be clinically useful in the detection of large episodes of FMH requiring maternal treatment with more than the standard dose of Rh-immune globulin, it is laborious and suffers from subjectivity and imprecision (7, 8). Besides the experience of the laboratory technicians for interpretation of the results, the test has a tendency to overestimate the size of feto-maternal hemorrhages because maternal HbF-containing RBCs or F-cells are counted within the population of fetal cells (9).
Several alternative and more accurate screening methods to detect FMH using flow cytometry have been proposed and described. The first reports investigating the feasibility of using flow cytometry for fetal cell counting primarily relied upon the detection of the human D antigen on the cell surface of RBCs (10, 11, 12, 13). These approaches all demonstrated greater sensitivity and precision than manual methods. However, the use of anti-RhD is applicable only to the clinical situations with Rh or D antigen incompatibility and cannot be utilized in all cases of maternal trauma and suspected FMH. Several other methods for flow cytometric detection of fetal cells in maternal peripheral blood have recently been described. The methods differ in their means of using various cellular fixation and permeabilization steps, usually in combination with the intracellular detection of fetal hemoglobin (HbF) antigen using anti-HbF antibodies.
As an increase of the expression of fetal hemoglobin (HbF) in peripheral red blood cells is also a common feature in hemoglobinopathies comprising genetic disorders of hemoglobin such as sickle-cell disease and beta-thalassemia (14, 15, 16, 17), a method for the detection of HbF in blood cells also finds its use in the diagnosis of hemoglobinopathies other than those related to FHM.